PCr Notes
1.
Joel prefers to use 10X buffer without Mg added so that it can be adjusted. He
uses a standard final concentration of 1.5mM Mg.
2.
Joel orders primers from GenSet (?) and prefers to have primers already
resuspended not in lyophilized form. Primers are used at a concentration of
100ng/ul.
3.
dNTP’s are made up in a cocktail so that the total final concentration is 10mM of
dNTP and so each NTP is at 2.5mM concentration.
4.
PCr reaction volume is 50ul. An example volume mixture is listed below:
H2O
10X buffer
MgCl2 (50mM)
DNTP’s (10mM)
5’ primer (50M)
3’ primer (50M)
template
Taq
29.25
5
1.5
(final concentration 1.5mM)
5
0.5
(final conc. is 0.5(range 0.2-1M)
0.5
(final conc. is 0.5(range 0.2-1M)
8
0.25
Make up enough for at least 1 tube more than the total number needed.
If running two sets of primers decrease the water and keep total primer
volume at 1ul. DMSO can be added if bands are weak or non-existant (110%)
Below is an example data sheet used (primers are denoted in top row):
Date
Sample
H2O
10X
Buffer
MgCl2
DNTP’s
LL507
5’tTA
GF100
3’tTA
PKCß
5’PKC
GF82
3’PKC
Template
Thermocycler is usually run at a 50°C annealing temperature.
PCr reaction is run on a 2% agarose gel for approximately 45 – 60 minutes. Product is
approximately 500bps.
Example thermocycler protocol
94°C 2 min/94°C 1min – 50° 30sec – 72° 30sec/ 72° 7 min/ 4°C infinite
Taq